During retinal degeneration, cone photoreceptors make ectopic synapses with rod bipolar cell dendrites. These data provided critical evidence of synaptic remodeling in the outer retina, with the further implication that alterations in cellular function may occur widely in the diseased retina. Defects in the regulation of calcium in the diseased retina may cause neuronal death as well as changes in neighboring cells that are synaptically connected to the affected cells. Therefore, defects in calcium regulation could cause wide-spread modifications of cellular function throughout the retina, and have a profound impact on the diseased retina. In a search for potential alterations in calcium regulation in diseased retinas, the PI identified one of the least well understood mechanisms, "store-operated calcium entry" (SOCaE) or "capacitative calcium entry" (CCE), in retinal ganglion cells (RGC) of the normal retina. Furthermore, preliminary observations strongly suggested that CCE in RGC of a murine model of retinal degeneration may be modified. The present proposal addresses the hypothesis that intracellular calcium regulation may be modified in RGC of animal models of retinal degeneration. The first aim of the study is to characterize one of the key calcium regulatory mechanisms, CCE, in normal murine and porcine RGC. The second aim is to define the alterations in CCE in RGC of diseased retinas. The research plan will entail: (1) establishing in vitro experimental protocols for studying retinal explants and RGC isolated from mice and pigs; (2) intracellular calcium measurements using ratiometric methods; (3) measuring the calcium-release activated calcium current (Icrac) in RGC isolated from normal and diseased retinas with patch-clamp recording techniques. Calcium release from internal stores and regulation of calcium levels are key events in nearly all cells. In degenerating retinas, the mechanisms and pathways involved could be significant for remodeling of neurons downstream of photoreceptors. The results of this research should elucidate some of the key pathways of intracellular calcium regulation in retinal neurons, and identify specific changes in calcium regulatory mechanisms that may occur in degenerating retinas.